Blunt end DNA ligation의 최적반응조건을 구하기 위하여 반응온도, 완충용액상태, vector와 insert DNA의 농도비율, 반응시간등의 최적조건을 연구하였다. 실험결과 반응최적온도는 $20-25^{circ}C$였고, ATP의 최적농도는 0.5 mM이었다. Spermidine의 농도가 1 mM 인 경우와 4%의 PEG 4,000을 ligation buffer에 넣어준 경 우 반응이 잘 되는 것으로 나타났으나 spermidine이 10 mM이거나 ATP의 농도가 2 mM 이상에서 반응은 저해되었다. 높은 농도의 NaCl 또한 반응을 저해시키는 것으로 나타났다. Vector와 insert DNA의 농도비를 달리하여 ligation시켜 이것을 직접 E. coli를 형질 전환시켜 본 결과 농도비가 1일때 insert가 끼어 들어간 확율이 가장 높았다. Buffer의 pH변화(6.6-8.2)나 BSA의 농도변화 ($0-500;{mu}g/ml$)는 반응에 별 영향이 미치지 않는 것으로 나타났다.
To determine the optimal condition of blunt-ended DNA ligation for the purpose of the efficient cloning, blunt-ended vector and insert DNA fragments were ligated under the various conditions of buffer, temperature, reaction time, and the molar ratio of vector to insert DNA. The efficiency of ligation was monitored by both agarose gel electrophoresis and the transforming frequency of genetic markers in plasmid pRSK 116 in vivo. The results showed that the optimal incubation temperature and the concentration of ATP were $20-25^{circ}C$ and 0.5 mM, respectively. Spermidine (1 mM) and polyethylene glycol (3%) stimulated blunt-end ligation, but more than 10 mM of spermidine and 2 mM of ATP completely inhibited the reaction. Higher concentration of salt also inhibited blunt-end ligation. According to the transforming frequency assay, the optimal molar ratio of vector to insert DNA was 1, which was different from that of sticky-end ligation. However, the variation of pH (6.6-8.2) and bovine serum albumin concentration ($0-500;{mu}g/ml$) showed little influence on the ligation.
